Background: Hepatitis C virus (HCV) non-structural protein 5A (NS5A) inhibitors have been recently developed to inhibit NS5A activities and have been approved for the treatment of HCV infection. However the drawback of these direct acting antivirals (DAAs) is the emergence of resistance mutations. The prevalence of such mutations conferring resistance to HCV-NS5A inhibitors before treatment has not been investigated so far in the Tunisian population. The aim of this study was to detect HCV variants resistant to HCV-NS5A inhibitors in hepatitis C patients infected with HCV genotype 1 before any treatment with NS5A inhibitors. Methods: Amplification and direct sequencing of the HCV NS5A region was carried out on 112 samples from 149 untreated patients. Results: In genotype 1a strains, amino acid substitutions conferring resistance to NS5A inhibitors (M28V) were detected in 1/7 (14.2 %) HCV NS5A sequences analyzed. In genotype 1b, resistance mutations in the NS5A region (R30Q; L31M; P58S and Y93H) were observed in 17/105 (16.2 %) HCV NS5A sequences analyzed. R30Q and Y93H (n = 6; 5.7 %) predominated over P58S (n = 4; 3.8 %) and L31M (n = 3; 2.8 %). Conclusions: Mutations conferring resistance to HCV NS5A inhibitors are frequent in treatment-naive Tunisian patients infected with HCV genotype 1b. Their influence in the context of DAA therapies has not been fully investigated and should be taken into consideration.